The development of antimicrobial agents led to a significant decrease in morbidity and mortality from infectious disease in this century. This accomplishment was largely due to the widespread use of the major classes of antibiotics, such as the sulfonamides, penicillins, cephalosporins, aminoglycosides, tetracyclines (Goodman et al., The Pharmacological Basis of Therapeutics, Macmillan Publishing, New York, 1985). However, in recent years, the trend in reducing infectious disease mortality has been threatened by the emergence of resistant strains of microorganisms that are no longer susceptible to the currently available antimicrobial agents. As a result, maintenance of public health requires that new antimicrobial agents be developed to counter these emerging resistant strains in order that diseases previously considered to be under control do not reemerge.
Recently, a group of peptides with antimicrobial properties has drawn attention as potential therapeutic agents. Such peptides include the magainins produced by amphibians and the cecropins produced by insects (Jacob et al., Ciba Foundation Symposium, John Wiley & Sons, New York, 1994). These peptides are believed to function as multimeric structures that insert into biological membranes to form pores that disrupt membrane structure and cause a loss of osmotic integrity. Although a wide sequence variation has been observed among these peptides, they all appear to retain a characteristic positively charged amphipathic helical structure, indicating that structural organization is a critical determinant of their membrane-disruptive functionality.
The magainins have been extensively investigated for clinical application, and have been found to be effective in treating systemic E. coli infections in a mouse model. Trials are underway to evaluate a magainin analog for effectiveness as a topical antimicrobial for the treatment of impetigo (Jacob et al., Antimicrobial Peptides, Ciba Foundation Symposium, 197-223, 1994).
Intensive investigation into the cytopathology caused by the human immunodeficiency virus (HIV) has included the structural analysis of its proteins. The HIV transmembrane (TM) protein (gp41), which is incorporated into the membrane of the virion, comprises three domains: an ectodomain, a transmembrane region and an intravirion endomain region, the latter comprising approximately 150 amino acids. The endodomain contains two regions in its carboxy terminus which have a high hydrophobic moment and are capable of forming amphipathic .alpha.-helices (Eisenberg et al., Biopolymers 29:171-177, 1990). This structural feature of the endodomain is similar to the three-dimensional structure of known cytolytic peptides, despite the lack of any amino acid sequence homology with them.
Miller et al. demonstrated that synthetic peptides corresponding to sequences found in the carboxy terminus of the TM protein of both HIV-1 and simian immunodeficiency virus (SIV), termed HIV-L and SIV-L, respectively, were capable of cytolytic activity against procaryotic and mammalian cells (Miller et al., AIDS Res. and Hum. Retro. 7:511-519, 1991). The term "LLP" was used to designate lytic peptides derived from lentiviruses (such as HIV and SIV). Other studies confirmed the antimicrobial activity of these peptides (Srinivas et al., J. Biol. Chem. 267:7121-7127, 1992; Arroyo et al., J. Virol. 69:4095-4102, 1995). Antifungal activity has also been demonstrated for a synthetic peptide corresponding to this region (Zhong et al., Int. J. Peptide Protein Res. 45:337-347, 1995). In contrast to other antimicrobial peptides which are specifically encoded by their own genes, LLPs are unique in that they are derived from naturally occurring sequences that are part of a larger folded protein.
Further analysis confirmed that LLPs could cause membrane perturbation in mammalian cells (Srinivas et al., J. Biol. Chem. 267:7121-7127, 1992; Miller et al., Virology 196:89-100, 1993), Sf9 cells (Chernomordik et al., J. Virol. 68:7115-7123, 1994) and E. coli (Arroyo et al., J. Virol. 69:4095-4102, 1995). Other studies demonstrated that synthetic peptides corresponding to a portion of the HIV TM protein also could inhibit the fusion of HIV-infected CD4+ cells (Srinivas et al., J. Biol. Chem. 267:7121-7127, 1992). Moreover, a similar synthetic peptide corresponding to the same region of gp41 directly interacted with the lipid bilayer of microsomal membranes (Gawrisch et al., Biochemistry 38:3112-3118, 1993) and caused pore formation in membranes (Chernomordik et al., J. Virol. 68:7115-7123, 1994).
In further studies designed to analyze the mechanism of HIV cytopathogenesis, synthetic peptides corresponding to regions of the HIV TM protein were shown to bind to calmodulin (CaM), a critical mediator of Ca2+-based signal transduction in eucaryotic cells (Miller et al., AIDS Res. and Hum. Retro. 9:1057-1066, 1993; Srinivas et al., J. Biol. Chem. 268:22895-22899, 1993). An analogous peptide from SIV was also shown to bind CaM (Yuan et al., Biochemistry 34:10690-10696, 1995). These findings imply that HIV and other lentiviruses may interfere with the cellular signalling pathways by sequestering CaM through a TM protein-mediated mechanism.
Certain analogs of LLP1 have been tested for activity in erythrocyte lysis assays and CaM binding assays (Tencza et al., J. Virol. 69:5199-5202, 1995). For example, analogs with a single amino acid replacement that produced a charge substitution, e.g., Arg.fwdarw.Glu, showed significantly decreased cytolytic potential and CaM binding ability. Analogs in which a hydrophobic residue was replaced by a non-hydrophobic amino acid, e.g., Ile.fwdarw.Ser, exhibited a significant reduction in cytolytic activity, but less reduction in the ability to bind CaM.
Analysis of gp41 from diverse HIV isolates from which LLP1 has been obtained reveals a 28-amino acid segment which exhibits a conservation of charge number and amphipathic potential, even though amino acid sequence variation exists in this region of gp41 from different virus isolates (Miller et al., Virology 196:89-100, 1993).
Examination of sequences from a variety of lentiviruses also revealed an LLP1 region in SIV and equine infectious anemia virus (EIAV) (Miller et al., AIDS Res. Hum. Retroviruses 7:511-519, 1991).
Another sequence motif was identified in the HIV-1 TM protein upstream of the LLP1 sequence (Eisenberg et al., Biopolymers 29:171-177,1990), and was designated LLP2. Similar sequences existing in the SIV TM protein have also been identified. The SIV protein contains an unusually long LLP2 region (amino acids 771-817 of SIV MM239). The LLP2 sequences among HIV-1 isolates are well conserved, having much less amino acid variation than seen in LLP1 sequences (Myers et al., Human Retroviruses and AIDS 1995, Los Alamos National Laboratory, 1995).